INTRACELLULAR LIFESTYLE OF PSEUDOMONAS AERUGINOSA

描述（由申请人提供）：铜绿假单胞菌可导致严重的危及视力和生命的疾病。上皮衬里的表面组织如眼睛、皮肤和气道是最常见的靶向部位。易感人群包括囊性纤维化儿童、免疫功能低下者、烧伤患者、插管患者和隐形透镜配戴者。铜绿假单胞菌感染的发病率正在上升;令人担忧的是，它往往具有高度破坏性，并与预后不良有关。众所周知，铜绿假单胞菌感染很难使用可用的疗法治疗，部分原因是铜绿假单胞菌拥有大量致力于生存和适应的基因。因此，迫切需要新的治疗方法。虽然已知铜绿假单胞菌可以在感染期间进入上皮细胞，并且细胞侵袭可以是发病机制的关键组成部分，但对铜绿假单胞菌在任何细胞类型内的细胞内生活方式知之甚少。本研究的目的是了解铜绿假单胞菌使用的细胞内生存策略，并确定它们是否可以在眼部感染模型中降低体内毒力。初步数据显示，铜绿假单胞菌在上皮细胞内占据了一个新的细胞内生态位;感染诱导的质膜水泡。在这些水泡中，细菌复制并表现出快速（实时可见）的运动性。这些数据表明，第三型分泌系统（T3 SS）是由铜绿假单胞菌形成水泡龛所必需的。T3 SS突变体不能形成泡，而是定位于核周空泡。与野生型细菌相反，T3 SS突变体（保留入侵能力）在进入上皮细胞后失去活力。T3 SS效应子和转运T3 SS效应子穿过宿主细胞膜所需的易位子都在铜绿假单胞菌细胞内存活/运输中起作用。效应子突变体和易位子突变体各自缺乏起泡能力和向核前液泡的运输，然而，只有效应子突变体失去细胞内复制的能力。因此，数据表明T3 SS在细胞内存活中至少有两种作用：1）核周空泡中效应子依赖性细胞内复制，和2）translocon依赖性水泡龛形成。待检验的假设是：目的1：在T3 SS效应物不存在的情况下，铜绿假单胞菌在溶酶体内降解，但特异性T3 SS效应物操纵内吞运输以使其能够在核周空泡中存活。目标二：T3 SS通过使细菌从空泡隔室中逃逸（translocon依赖性）参与水泡生态位的形成，并且T3 SS在水泡形成/向其运输中也有直接作用。目的3：体内细胞内存活也是T3 SS依赖的，并且可以靶向操纵毒力。目的1和2将涉及体外细胞培养感染方法、细菌突变体、活力测定和使用和不使用分子事件抑制剂/激活剂的成像。目标3将使用完善的体内角膜感染模型、目标1和2的方法以及细菌定植和疾病严重程度的定量来完成。公共卫生相关性：由铜绿假单胞菌引起的感染通常与不良预后相关，因为这种病原体固有地对杀灭具有抗性，可能具有高度破坏性，并且易感人群包括已经因现有疾病如囊性纤维化、免疫功能低下、烧伤或其他损伤而虚弱的人。铜绿假单胞菌在感染期间可变为细胞内的，并且这已被证明有助于体内发病。虽然已经研究了细胞进入机制，但几乎不知道铜绿假单胞菌在侵入后在细胞内存活的机制。研究计划的重点是研究铜绿假单胞菌在细胞内生存的策略，然后确定它们作为新疗法靶点的潜力。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa can cause severe sight- and life-threatening disease. Epithelial lined surface tissues such as the eye, the skin and the airways are the most commonly targeted sites. Susceptible populations include children with cystic fibrosis, immunocompromised individuals, burn victims, intubated patients, and contact lens wearers. The incidence of P. aeruginosa infection is rising; worrisome given that it is often highly destructive and associated with a poor prognosis. P. aeruginosa infection is notoriously difficult to treat using available therapies, in part because P. aeruginosa possesses a large number of genes devoted to survival and adaptation. Thus, new approaches to therapy are urgently needed. While it is known that P. aeruginosa can enter epithelial cells during infection, and that cell invasion can be a key component in pathogenesis, little is known about the intracellular lifestyle of P. aeruginosa within any cell type. The objective of this research is to understand intracellular survival strategies used by P. aeruginosa and to determine if they can be targeted to reduce virulence in vivo in an eye infection model. Preliminary data reveal that P. aeruginosa occupies a novel intracellular niche within epithelial cells; infection-induced plasma membrane blebs. In these blebs, bacteria replicate and demonstrate rapid (real-time visible) motility. The data show that the Type Three Secretion System (T3SS) is required for bleb-niche formation by P. aeruginosa. T3SS mutants fail to form blebs and instead localize to perinuclear vacuoles. In contrast to wild type bacteria, T3SS mutants (retain competency for invasion) lose viability after entering epithelial cells. The T3SS effectors and the translocon required for transporting T3SS effectors across host cell membranes both play roles in P. aeruginosa intracellular survival/trafficking. Effector mutants and translocon mutants each lack blebbing capacity and traffic to preinuclear vacuoles, however, only effector mutants lose capacity for intracellular replication. Thus, the data suggest at least two roles for the T3SS in intracellular survival; 1) effector-dependent intracellular replication in perinuclear vacuoles, and 2) translocon-dependent bleb niche formation. The hypotheses to be tested are: Aim 1: That in the absence of T3SS effectors, P. aeruginosa is degraded within lysosomes, but specific T3SS effectors manipulate endocytic trafficking to enable survival in perinculear vacuoles. Aim 2: That the T3SS participates in bleb-niche formation by enabling bacterial escape from vacuolar compartments (translocon-dependent) and that there are also direct roles for the T3SS in bleb formation/trafficking to them. Aim 3: That intracellular survival in vivo is also T3SS-dependent, and can be targeted to manipulate virulence. Aims 1 and 2 will involve in vitro cell culture infection methods, bacterial mutants, viability assays and imaging used with and without inhibitors/activators of molecular events. Aim 3 will be done using a well-established in vivo corneal infection model, methods from aims 1 and 2, and quantification of bacterial colonization and of disease severity. PUBLIC HEALTH RELEVANCE: Infections caused by Pseudomonas aeruginosa are often associated with a poor prognosis because this pathogen is inherently resistant to killing, can be highly destructive, and susceptible populations include people already debilitated by existing conditions such as cystic fibrosis, immunocompromise, burns or other injury. P. aeruginosa can become intracellular during infection, and this has been shown to contribute to pathogenesis in vivo. While cellular entry mechanisms have been studied, almost nothing is known about mechanisms used by P. aeruginosa for survival within cells after invasion. The focus of the research plan is to study strategies used by P. aeruginosa for surviving intracellularly and then to determine their potential as targets for new therapies.